Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

Abstract

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Figure 1. ATPS-ELISA prevents polyclonal detection antibody cross-reactions.

Multiplex ATPS-ELISA (a) and conventional sandwich ELISA (b) share similar procedures, shown in steps i., ii., and iii. However, by co-localizing detection antibodies (dashed line antibody symbols) in the DEX phase over the corresponding capture antibodies (solid line antibody symbols) through the use of simple micropipetting onto custom plates with features designed for micropipette tip alignment, ATPS-ELISA produces signals without any possibility of cross-reactions. (c) Goat detection antibodies were not captured by a neighboring spot coated with an anti-goat capture antibody, indicating that antibodies did not diffuse out of the DEX droplet to cause cross-reactions. (d) Bath application of detection antibodies resulted in cross-reactions at the neighboring capture antibody spot.

Figure 2. DEX droplets assume dome shapes that change very little in size and shape over the course of the incubation period.

(a) Antibodies are retained in the DEX domes over this period as indicated by the overlap between FITC-DEX and PE-IgG. Scale bar = 1 mm. (b) Biotin-labeled ELISA detection antibodies partition favorable to the DEX phase. Partition coefficients were measured by blotting detection antibody fractions from PEG and DEX on PVDF membranes and detecting the antibody levels by way of streptavidin-HRP chemiluminescence. Partitioning can be further improved by modifying ATPS formulations. (c) Partial overlap of the capture and detection antibodies results in a cat eye shape that can only be produced if antibodies are well retained in the DEX droplet. (d) Bath application of detection antibodies produced a circular signal area.

Figure 3. ATPS ELISA can be used to pattern 9-plex and 16-plex arrays of detection antibodies.

(a) A 9-plex version of the experiment shown in Figure 1 c that demonstrates how ATPS-ELISA suppresses cross-reactions. Goat anti-ST2 detection antibody solutions are dispensed as ATPS droplets only to the 5 regions with spotted ST2 capture antibodies and ST2 antigen. These 5 regions generated true-positive signals. The 4 other regions spotted with anti-goat capture antibodies that would cross-react and give false positive signals with the goat anti-ST2 detection antibody did not receive any detection antibody solution droplets and thus resulted in no signal, demonstrating suppression of false positive signals. (b) A 9-plex version of the experiment shown in Figure 1 d. Bath applied goat anti-ST2 detection antibodies become sequestered by the 4 anti-goat antibody spots. This produces 4 false positive readouts and interestingly 5 false negative signals with very low (but detectable) signal levels because there is insufficient ST2 detection antibody available to bind to the ST2 sandwich regions despite the fact that the anti-ST2 capture antibodies are bound to ST2. (c) Representative image of a 16-plex ATPS sandwich ELISA for ST2 with bath application of the capture antibody and localized dispensing of detection antibody in DEX droplets. (d) Same experiment as in c but without localized dispensing of detection antibody in the DEX droplets. The result is a signal from the entire well rather than localized signals.

Figure 4. Multiplex ATPS-ELISA for GVHD biomarkers.

(a) A 3D rendering showing the custom plate design consisting of 4 antibody insets within a common shallow sample well. (b) Representative images of the chemiluminescent standards. The concentrations listed above each image are the same for each biomarker in the panel. The images correspond to every third point on the quarter-logarithmic dilution curve from 10,000 pg/mL to zero. In each of the images the spot at the top corresponds to HGF, the spot on the right corresponds to elafin, the spot at the bottom corresponds to ST2 and the spot on the left corresponds to TNFR1. It is apparent from these images that elafin has the highest limit of detection due to its high background (elafin can be detected in healthy plasma as well as in GVHD+ plasma). Since the biomarker standards contained a 10% healthy pooled plasma to adjust for matrix affects (see “Plasma samples” in the Methods section), this background can be attributed to baselines levels of elafin and should not be confused as an example of a false positive signal. (c–e) Standard curves for all four GVHD biomarkers in PBS generated by densitometric quantification of chemiluminescence images compared to individual sandwich ELISAs (dashed lines). Error bars represent standard error of the mean.

Figure 5. Multiplex ATPS-ELISA enables robust detection of GVHD biomarkers in patient plasma samples.

The ATPS-ELISA multiplex detection system was used to probe human plasma for four biomarkers: (a) HGF, (b) elafin, (c) ST2 and (d) TNFR1. The GVHD+ group displayed significantly higher levels of all four biomarkers compared to the GVHD – and healthy control groups (p<0.05 by one-way ANOVA with Dunn's multiple comparison test). Measurements of (e) HGF, (f) elafin, (g) ST2 and (h) TNFR1 from patient plasma using individual sandwich ELISAs are provided for comparisons. Significance between the GVHD+ group and the GVHD- and healthy control groups was obtained for HGF and TNFR1 (p<0.05 by one-way ANOVA with Dunn's multiple comparison test) for individual ELISAs, but not for elafin or ST2, although ST2 values for the GVHD+ group tended to be higher than the other groups. Error bars represent standard error of the mean.

Figure 6. Bland-Altman analysis for (a) HGF, (b) elafin, (c) ST2 and (d) TNFR1.

The dashed horizontal lines represent the 2SD confidence intervals and the solid horizontal line represents the mean difference between assay formats. We observed discrepancies between the two assay formats, particularly at the higher biomarker concentrations.